Blood glucose is an important marker for diabetes. In the fermentative production using microorganisms, glucose levels are assayed for monitoring the process. Conventional glucose assays were based on enzymatic methods using glucose oxidase (GOD) or glucose-6-phosphate dehydrogenase (G6PDH). However, GOD-based assays require addition of catalase or peroxidase to the assay system in order to quantitate the hydrogen peroxide generated by glucose oxidation reaction. G6PDHs have been used for spectrophotometric glucose assays, in which case a coenzyme NAD(P) must be added to the reaction system.
Recently, PQQGDH is used as a new enzyme in place of GOD or G6PDH, which have been conveniently used for enzymatic assay of glucose (see, JP A 10-243786, WO 00/66744, WO 00/61730). Since PQQGDH exhibits a high oxidation activity for glucose and is a coenzyme-associated enzyme that does not require oxygen as an electron acceptor, it is useful as a recognition element of a glucose sensor.
PQQGDHs catalyze the reaction in which glucose is oxidized to produce gluconolactone. PQQGDHs include membrane-bound enzymes and water-soluble enzymes. Membrane-bound PQQGDHs are single peptide proteins having a molecular weight of about 87 kDa and widely found in various gram-negative bacteria. Water-soluble PQQGDHs have been identified in several strains of Acinetobacter calcoaceticus (Biosci. Biotech. Biochem. (1995), 59(8), 1548-1555), and their structural genes were cloned to show the amino acid sequences (Mol. Gen. Genet. (1989), 217:430-436). The water-soluble PQQGDH derived from A. calcoaceticus is a homodimer consisting of two subunits having a molecular weight of about 50 kDa. Each subunit has one PQQ molecule and three Ca++ cations. The dimer protein has two active centers, while the monomer enzyme does not have a GDH activity. Thus, formation of a dimer structure is essential for the GDH activity. The X-ray analysis of the crystalline structure of the water-soluble PQQGDH revealed the higher-order structure of the enzyme (J. Mol. Biol., 289, 319-333 (1999), A. Oubrie et al., EMBO Journal, 18(19), 5187-5194 (1999), A. Oubrie et al., PNAS, 96(21), 11787-11791 (1999), A. Oubrie et al.).
The object of the present invention is to provide a modified water-soluble PQQGDH having improved thermal stability.